The overall objective of this proposal is to delineate mechanisms of clonal anergy and deletion noted in transplant (Tx) protocols. Using a sensitive, reproducible limiting dilution analysis (LDA) technique, evidence was obtained of a donor-specific regulatory cell that inhibits CTL precursors (CTLp) in long-term renal Tx recipients receiving conventional immunosuppression and in recipients who received pre-Tx donor specific transfusions (DST). In addition, many long-term DST- treated (DST-A) renal allograft recipients demonstrate specific deletion of anti-donor CTLp. to learn whether clonal deletion of donor-reactive CTLp and/or the development of donor-specific suppression contribute to the state of allograft tolerance, we will perform time course studies to follow changes in CTLp with specificity for donor and third party antigens in DST-A and non-DST-A allograft recipients, thereby determining if clonal reduction occurs more rapidly, to a greater extent and with more frequency in DST vs non-DST patients. Moreover, we will evaluate the relationship between donor-specific hyporesponsiveness, deletion of donor-reactive CTLp and the emergence of regulatory cells. Results will be analyzed for correlation with recipient's HLA phenotype, HLA matching with donor, immunosuppressive therapy, renal function, rejection episodes, and long-term graft outcome. If suppression is mediated by a regulatory T cell, we will clone it and test the hypothesis that it is a self-restricted CTL which recognizes donor-specific allopeptides bound to self-HLA. We will determine whether the apparent clonal deletion of anti-donor CTLp represents actual clonal elimination or clonal anergy. If clonal anergy is established as the mechanism, we will determine how it influences CTL responsiveness. Or to prove true deletion of anti- donor CTLp, we will characterize and estimate by semi-quantitative PCR the V gene families present in CD8+ T cells of patient PBL pre-A, post-A, and post-Tx, and look for correlation between elimination of anti-donor alloreactivity and deletion of specific V elements from the repertoire. Anergy/deletion of donor-reactive CTLp may not sufficiently explain tolerance to donor antigens in renal allograft recipients. Therefore, we will evaluate the contribution to acquired immune tolerance of anti- clonotypic antibodies and alterations in the TH cell compartment (e.g., a predominance of TH2 over TH1). Finally, we will determine the immunologic relevance of a CD3/TCR cell population which we observe in DST-A patients. We will probe the origin, phenotype, and functional activities of these cells in order to evaluate if they are recipient's donor-reactive T cells in which down-regulation of CD3/TCR has occurred, or whether they are veto cells of donor origin. These studies on renal allograft recipients provide a unique opportunity to determine which of several immunological mechanisms implicated in animal models operate in human Tx tolerance, and whether they can be associated in a predictable manner with clinical course.